Monomeric self-associating fusion polypeptides and therapeutic uses thereof

ABSTRACT

Monomeric fusion polypeptides comprising R1-M-L-M, wherein R1 is a target ligand-binding domain, M is a multimerizing component, and L is a linker capable of allowing one M component to interact with the other M component to form a self-associating monomer, optionally comprising a second target ligand-binding domain R2. Preferred target ligands include interleukin (IL)-13, IL-1, insulin-like growth factor (IGF)-1 and IGF-2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 USC §119(e) of U.S. Provisional 60/728,255 filed 19 Oct. 2005, which application is herein specifically incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention encompasses receptor-based fusion polypeptides, as well as therapeutic uses of such polypeptides. More specifically, the invention features monomeric fusion polypeptides comprising receptor components and self-associating components.

2. Description of Related Art

In U.S. Pat. No. 6,472,179 Stahl et al. describe cytokine fusion protein fusion polypeptides capable of binding a cytokine to form a nonfunctional complex composed of two receptor components and a multimerizing component. The interleukin-13 receptor alpha component (IL-13Rα) is described, e.g., U.S. Pat. Nos. 5,710,023 and 6,248,714 (Collins et al.), which publications are herein incorporated by reference in their entireties.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention features a nucleic acid molecule encoding a monomeric, self-associating fusion polypeptide R1-M-L-M, wherein R1 is a target ligand-binding domain, M is a self-associating component, and L is a linker.

R1 may be one or more receptor components defining a ligand-binding domain. In a preferred embodiment, R1 is a single receptor component capable of exhibiting high affinity binding to a target ligand. In one embodiment, the receptor component binds insulin-like growth factor (IGF)-1 or IGF-2. In a preferred embodiment, the receptor component binds a cytokine selected from one of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-30, IL-31, granulocyte macrophage colony stimulating factor, oncostatin M, G-CSF, GH, IGF-1, and leukemia inhibitory factor. In additional embodiments of the invention, the receptor component binds a cytokine which is a member of the interferon family of cytokines selected from the group consisting of IFN-gamma (IFN-γ), IFN-α, IFN-β, IL-28 and IL-29. In still further embodiments of the invention, the receptor component is capable of binding a cytokine which is a member of the TNF family of cytokines selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, RANKL, CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1BBL. In preferred embodiments, the receptor component binds a cytokine selected from the group consisting of IL-1, IL-10, IL-12, IL-14, IL-18, and MIF. In another embodiment the fusion partner is a ligand, such as EPO, TPO, G-SCF, GM-CSF, IL-1ra, CNTF, Axokine, GLP-1, IFNβ, IFN-A2, GH, IFG-1, IGF-2, IL-2, IFN-γ, IL-4, IL-21, IL-24, KGF, PYY, thrombin, etc.

In one embodiment of the invention, the nucleic acid molecule further encodes a second target-ligand binding domain (R2), wherein R2 is specific for the same or a different target ligand-binding component as R1. In one embodiment, the components are arranged as R1-M-L-M-R2. In another embodiment, the components are arranged as R1-R2-M-L-M.

The self-associating component (M) is any component that is capable of interacting with the second M component on the same fusion polypeptide, that is, forming an M:M structure. Preferably, the M component enhances the functionality of the fusion polypeptide. Thus, for example, a self-associating component may enhance the biological activity of the fusion polypeptide, aid in its production and/or recovery, or enhance a pharmacological property or the pharmacokinetic profile of the fusion polypeptide by, for example, enhancing its serum half-life, tissue penetrability, lack of immunogenicity, or stability.

M may be any natural or synthetic sequence capable of interacting with another self-associating component. The two M components of the fusion polypeptide of the invention will generally be the same. In specific embodiments, the self-associating component is selected from the group consisting of (i) an immunoglobulin-derived domain, (ii) an amino acid sequence between 1 to about 500 amino acids in length, comprising at least one cysteine residue, (iii) a leucine zipper, (iv) a helix loop motif, and (v) a coil-coil motif. In a more specific embodiment, the immunoglobulin-derived domain is selected from the group consisting of the Fc domain of IgG (IgG₁, IgG₂, IgG₃ or IgG₄) or the heavy chain of IgG. In one embodiment, the Fc domain of IgG is human FcΔ1(a), an Fc molecule with a mutation of the region involved in forming the disulfide bond with the light chain.

The linker L is a component which allows the multiple M components to form an intra-molecular interaction, e.g., a self-associating monomer. L may be a sequence between about 10 to about 30 amino acids in length. In a preferred embodiment, L is a sequence about 16 amino acids in length, for example the peptide of SEQ ID NO:2. In another embodiment, L is the G4S linker of SEQ ID NO:3.

The nucleic acid molecule of the invention may further optionally comprise a signal sequence (SS) component. When a SS is part of the polypeptide, any SS known to the art may be used, including synthetic or natural sequences from any source, for example, from a secreted or membrane bound protein. In one preferred embodiment, an ROR signal sequence is used (SEQ ID NO:4).

In one specific embodiment, the nucleic acid of the invention encodes a fusion protein R1-M-L-M, wherein R1 is an IL-13 receptor alpha (IL-13Rα2), M is an Fc, L is a peptide between 10-20 amino acids, and the self-associating monomeric fusion protein is capable of specifically inhibiting IL-13 activity with an IC₅₀ of at least 10⁻¹⁰ molar. More specifically, R1 is an IL-13Rα2 component amino acids 1-343 or 23-343 of SEQ ID NO:1, optionally modified with one or more of the modifications defined in modification group I.

Modification Group I: (a) amino acids 1-22 of SEQ ID NO:1 are deleted. In specific embodiments in which it may be desirable to replace the deleted amino acids with, for example, a signal sequence such as SEQ ID NO:4, thus removing Cys22 to reduce aberrant disulfide bonds formation; (b) Cys252Ile of SEQ ID NO:1; (c) an amino acid changed at position 310 of SEQ ID NO:1. In a specific embodiment, Ser310 is replaced with Cys, which may be desirable to stabilize the tertiary structure of the protein.

In another embodiment, the nucleic acid encodes a fusion protein R1-M-L-M-R2, wherein R1 is the extracellular domain of IL-1R1 and R2 is the extracellular domain of IL-1RAcP. More specifically, R1 is SEQ ID NO:13 or a fragment thereof capable of binding IL-1 and R2 is amino acids 19-333 or 1-358 of SEQ ID NO:14, and the monomeric fusion polypeptide is capable of specifically inhibiting IL-1β activity with an IC₅₀ of at least 10⁻¹⁰ molar.

In another embodiment, the nucleic acid encodes a fusion protein R1-M-L-M-R2, wherein R1 and R2 are the fragments of the extracellular domain of hIGFR. More specifically, R1 is amino acids 1-489 and R2 is amino acids 721-736 of SEQ ID NO:6, and the monomeric fusion polypeptide is capable of specifically inhibiting IGF activity with a Kd of at least 10⁻⁹ molar.

In a related second aspect, the invention features a vector comprising a nucleic acid molecule of the invention. In further third and fourth aspects, the invention encompasses vectors comprising the nucleic acid molecules of the invention, including expression vectors comprising the nucleic acid molecules operatively linked to an expression control sequence, and host-vector systems for the production of a fusion polypeptide which comprise the expression vector, in a suitable host cell; host-vector systems, wherein the suitable host cell is, without limitation, a bacterial, yeast, insect, mammalian cell or plants, such as tobacco, or animals such as cows, mice, or rabbits. Examples of suitable cells include E. coli, B. subtilis, BHK, COS and CHO cells. Additionally encompassed are fusion polypeptides of the invention modified by acetylation or pegylation.

In a related fifth aspect, the invention features a method of producing a fusion polypeptide of the invention, comprising culturing a host cell transfected with a vector comprising a nucleic acid molecule of the invention, under conditions suitable for expression of the fusion polypeptide from the host cell, and recovering the polypeptide so produced.

In sixth, seventh, and eighth aspects, the invention features a fusion polypeptide comprising R1-M-L-M, wherein R1, M and L are as defined above, optionally further comprising R2, as defined above. The fusion polypeptide of the invention forms an intramolecular self-association between the two M components, which association is facilitated by the L component. In a preferred embodiment, the self-associating monomeric fusion polypeptide of the invention allows R to be active, whereas an R-M alone is not, e.g., when R is IL-13Rα2. In another embodiment, the self-associating monomeric fusion polypeptide of the invention is active as an antagonist capable of binding and inhibiting a target molecule and exhibited desirable properties relative to larger prior art molecules. For example, when R1 is the extracellular domain of IL-1Racp and R2 is the extracellular domain of IL-1R1, the fusion protein R1-M-L-R2 forms a self-associating protein capable of binding and inhibiting interleukin-1 (IL-1).

In a ninth aspect, the invention features pharmaceutical compositions comprising a fusion polypeptide of the invention with a pharmaceutically acceptable carrier. Such pharmaceutical compositions may comprise a monomeric polypeptide, or nucleic acids encoding the fusion polypeptide.

The monomeric, self-associating fusion polypeptides of the invention are therapeutically useful for treating any disease or condition, which is improved, ameliorated, or inhibited by removal, inhibition, or reduction of a target ligand. For example, when the monomeric fusion polypeptide of the invention is capable of binding IL-13, the monomeric fusion polypeptide is particularly useful for the treatment of asthma, which are improved, ameliorated, or inhibited by removal, inhibition, or reduction of IL-13. Accordingly, in a further aspect, the invention features a therapeutic method for the treatment of an IL-13-related disease or condition, comprising administering a fusion polypeptide of the invention to a subject suffering from an IL-13-related disease or condition. When the monomeric, self-associating fusion polypeptide is capable of binding IL-1, the invention features a therapeutic method for the treatment of an IL-1-related condition or disease, including for example, autoinflammatory diseases such as including familial mediterranean fever (FMF), NOMID/CINCA, Muckle-Wells Syndrome, FCAS, and tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS). When the monomeric, self-associating fusion polypeptide is capable of binding IGF-1 and/or IGF-2, the invention features a therapeutic method for the treatment of the IGF related conditions or disease, including for example, cancer or diabetic retinopathy. Although any mammal can be treated by the therapeutic methods of the invention, the subject is preferably a human patient suffering from or at risk of suffering from a condition or disease which can be improved, ameliorated, inhibited or treated with a fusion polypeptide of the invention.

In a further aspect, the invention further features diagnostic and prognostic methods, as well as kits for detecting, quantifying, and/or monitoring a target molecule with the monomeric fusion polypeptides of the invention.

Other objects and advantages will become apparent from a review of the ensuing detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods are described, it is to be understood that this invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.

Definitions

The term “high affinity for” a ligand means that the fusion polypeptide of the invention binds the intended ligand with an affinity of at least 10⁻⁹ molar, preferably at least 10⁻¹⁰ molar, and most preferably at least 10⁻¹¹ molar as determined by assay methods known in the art, for example, surface plasmon resonance analysis (e.g., BiaCore™). The term “capable of specifically blocking” or “capable of inhibiting the activity of” a target ligand means the fusion polypeptides of the invention inhibit the biological activity of the target ligand, as measured by bioassay. For example, when the target ligand is IL-13, bioassays may include luciferase-based assays using an STAT6 promoter element, and/or IL-13 stimulation of cell lines such as TF1 or of human peripheral blood cells with readouts such as growth or sCD23 secretion. When the target ligand is IL-1, bioassays may include luciferase-based assays using a NFKB promoter element, or IL-1 induced secretion of IL-6 by MRC5 cells. When the target ligand is IGF, bioassays include MCF-7 cell growth or AKT phosphorylation or binding assays (such as ELISA). “IC₅₀” is defined as the concentration of fusion protein required to inhibit 50% of the response to the target ligand as measured in a bioassay. The fusion polypeptides of the invention are preferably capable of inhibiting the biological activity of the target ligand with an IC50 or Kd of at least 1×10⁻⁹ M, preferably 10⁻¹⁰ M, even more preferably 10⁻¹¹ M.

A self-associating monomeric fusion polypeptide is a polypeptide possessing two associated components which form an intramolecular dimer. Thus the fusion polypeptides of the invention tend to form monomers rather than the dimers of the prior art (see, for example, U.S. Pat. No. 6,472,179 Stahl et al.).

The terms “treatment”, “treating”, and the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease, condition, or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for a disease or condition and/or adverse effect attributable to the disease or condition. “Treatment” as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; or (c) relieving the disease or condition, i.e., causing regression of the disease or condition. The population of subjects treated by the method of the invention includes subjects suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.

By the term “therapeutically effective dose” is meant a dose that produces the desired effect for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

As used herein, a “condition or disease” generally encompasses a condition of a mammalian host, particularly a human host, which is undesirable and/or injurious to the host. Thus, treating a condition or disorder with an IL-13-specific fusion polypeptide, an IL-1-specific fusion polypeptide, or an IGF-specific fusion polypeptide, will encompass the treatment of a mammal, in particular, a human, who has symptoms reflective of elevated or deleterious IL-13, IL-1, or IGF, or who is expected to have such decreased activation in response to a disease, condition or treatment regimen. Treating an IL-13-related, IL-1, or IGF-related condition or disease encompasses the treatment of a human subject wherein reducing IL-13, IL-1, or IGF levels with the fusion polypeptide of the invention results in amelioration of an undesirable symptom resulting from the IL-13-related, IL-1-related or IGF-related condition or disease.

General Description

The present invention provides fusion polypeptides capable of binding a target ligand with high affinity (e.g., a Kd of at least 10⁻⁹ M) which contain two self-associating multimerizing components and form a monomeric self-dimerized polypeptide. The fusion polypeptides of the invention offer several important advantages relative to fusion polypeptides containing a single multimerizing component. For example, when the self-associating component is Fc, the monomeric fusion polypeptides of the invention retain the activity of an Fc molecule but are smaller in size than a dimer formed by two R-Fc fusion. Such monomeric fusion polypeptides may exhibit better tissue penetration, ease of production and ease of dosage formulation. Monomeric fusion polypeptides are particularly advantageous when R is human IL-13Rα2 because the IL-13Rα2-Fc dimer is inactive, whereas the inventors have found that the self-associated IL-13Rα2-Fc-L-Fc monomer is capable of binding IL-13 with high affinity, as shown below. Similarly, monomeric versions of the IL-1 trap are low affinity inhibitors of IL-1, whereas the self-associating monomer is a high affinity inhibitor of IL-1β.

Nucleic Acid Constructs and Expression

The present invention provides for the construction of nucleic acid molecules self-associating monomeric polypeptides capable of binding a target ligand with high affinity, e.g., a Kd of at least 10⁻⁸ M. As described above, the nucleic acid molecules of the invention encode self-associating fusion polypeptides capable of binding a target ligand with high affinity. Accordingly, the nucleic acid molecules may be termed “recombinant”, “artificial”, or “synthetic” as they are not nucleic acid molecules found in nature, e.g., not naturally occurring sequences, but are sequences constructed by recombinant DNA technology.

These nucleic acid molecules are inserted into a vector that is able to express the fusion polypeptides of the invention when introduced into an appropriate host cell. Appropriate host cells include, but are not limited to, bacterial, yeast, insect, and mammalian cells. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding the fusion polypeptides of the invention under control of transcriptional and/or translational control signals.

Expression of the nucleic acid molecules of the invention may be regulated by a second nucleic acid sequence so that the molecule is expressed in a host transformed with the recombinant DNA molecule. For example, expression may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression of the chimeric polypeptide molecules include, but are not limited to, a long terminal repeat (Squinto et al. (1991) Cell 65:1-20); SV40 early promoter region, CMV, M-MuLV, thymidine kinase promoter, the regulatory sequences of the metallothionine gene; prokaryotic expression vectors such as the beta-lactamase promoter, or the tac promoter (see also Scientific American (1980) 242:74-94); promoter elements from yeast or other fungi such as Gal 4 promoter, ADH, PGK, alkaline phosphatase, and tissue-specific transcriptional control regions derived from genes such as elastase 1.

Expression vectors capable of being replicated in a bacterial or eukaryotic host comprising the nucleic acid molecules of the invention are used to transfect the host and thereby direct expression of such nucleic acids to produce the fusion polypeptides of the invention. Transfected cells may transiently or, preferably, constitutively and permanently express the polypeptides of the invention.

Self-Associating Components

The fusion polypeptides of the invention comprise two self-associating components (M) separated by a linker (L) which allows the in-line multimerizing components to interact intramolecularly. M may be any natural or synthetic sequence capable of interacting with another associating component to form a self-associating monomer. The self-associating component may be selected from the group consisting of an immunoglobulin-derived domain, a truncated multimerizing component, an amino acid sequence between 1 to about 500 amino acids in length, a leucine zipper, a helix loop motif, and a coil-coil motif. When M is a self-associating component comprising an amino acid sequence between 1 to about 500 amino acids in length, the sequence may contain one or more cysteine residues capable of forming a disulfide bond with a corresponding cysteine residue on another fusion polypeptide comprising an M with one or more cysteine residues.

In a preferred embodiment, the self-associating component comprises one or more immunoglobulin-derived domain from human IgG, IgM or IgA. In specific embodiments, the immunoglobulin-derived domain is selected from the group consisting of the Fc domain of IgG or the heavy chain of IgG. The Fc domain of IgG may be selected from the isotypes IgG₁, IgG₂, IgG₃, and IgG₄, as well as any allotype within each isotype group. In one specific embodiment, M is the Fc domain of IgG₄ with Ser 228 (Cabot numbering) mutated to Pro to stabilize covalent dimer formation (Mol. Immunol. (1993) 30:105-108) and/or Leu235→Glu which eliminates residual effector functions (Reddy et al. (2000) J. Immunol. 164:1925-1933). In a preferred embodiment, M is the Fc domain of IgG₁, or a derivative thereof which may be modified for specifically desired properties (see, for example, Armour et al. (2003) Mol. Immunol. 40:585-593; Shields et al. (2001) J. Biol. Chem. 276:6591-6604).

Linker Components

The linker L is a component which allows the multimerizing components to form an intra-molecular interaction, e.g., a self-associating (self-multimerized) monomer. Although L may be any component which functions as required above, L is preferably an amino acid sequence between about 10 to about 30 amino acids in length. In a preferred embodiment, L is a sequence about 16 amino acids in length, for example the CT peptide (CTP) of SEQ ID NO:2. In another embodiment, L is the G₄S linker of SEQ ID NO:3. Other linkers may known to the art may be used, see, for example, George et al. (2003) Protein Engineering 15:871-879, herein specifically incorporated by reference.

Therapeutic Uses

The fusion polypeptides of the invention are therapeutically useful for treating any disease or condition which is improved, ameliorated, inhibited or prevented by removal, inhibition, or reduction of a target ligand. For example, in one embodiment the fusion polypeptide of the invention are capable of binding and inhibiting IL-13. IL-13 has been implicated in a variety of clinical conditions, such as eosinophil infiltration, IgE production and IL-5 production, that are characterized by a Th2 cell-driven response. Accordingly, the blocking of this responses by the fusion polypeptide will be useful for the treatment of any disease or condition in which there is increased occurrence of T-helper cells of the TH2 type.

In one embodiment, the IL-13-specific monomer of the invention is used to treat asthma. Data derived from animal experiments and examination of asthmatic humans implicate IL-13 as an initiator of the atopic condition and perpetuators of the chronic inflammatory state that typifies the asthmatic lung. IL-13 induces effects that are associated with the asthmatic phenotype, including isotype switching to IgE production, eosinophilia, mastocytosis, mucus formation, increased vascular permeability, airway hyper-responsiveness, smooth muscle hyperplasia, and subepithelial fibrosis (Hogan et al. (1997) Pharmcol. Ther. 74(3):259-283; McKenzie et al. (2000) Pharmacol. Ther. 88(2):143-151; Wills-Karp (2001) J. Allergy Clin. Immunol. 107(1):9-18).

A non-exhaustive list of specific conditions improved by inhibition or reduction of IL-13 include asthma, atopic dermatitis, immune complex disease (such as lupus, nephritis, and Graves' disease) allergic conditions, hyper IgE syndrome, immune deficiencies, idiopathic pulmonary fibrosis, hepatic fibrosis, HIV, pulmonary ‘remodeling,’ COPD, ulcerative colitis, cancer, Hodgkin's Lymphoma, cystic fibrosis, septic/reactive arthritis, ulcers, gastric inflammation, mucosal inflammation, Crohn's Disease, inflammatory bowel disease, dermatitis herpetiformis, chronic idiopathic urticaria, scleroderma, hypertrophic scarring, Whipple's Disease, benign prostate hyperplasia, lung allergic reactions to medication, Kawasaki disease, sickle cell disease (including sickle cell crisis), Churg-Strauss syndrome, pre-eclampsia, Sjögren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, tuberculosis, and nephrosis, adjuvants to allergy immunotherapy and as vaccine adjuvants, and myasthenia gravis, bullous pemphigoid, transplant and graft vs host disease, viral, parasitic, bacterial disease, and fungal infection. (U.S. Pat. No. 6,328,954; Idzerda, R. J. et al. 1990 J Exp. Med. 171:861-873).

The invention further provides a monomeric, self-associating fusion protein capable of binding interleukin-1 (IL-1) with high affinity. Conditions effectively treated by an IL-1 inhibitor or pharmaceutical composition described herein include pulmonary diseases such as asthma, chronic obstructive pulmonary disease, pulmonary alveolar proteinosis, bleomycin-induced pneumopathy and fibrosis, radiation-induced pulmonary fibrosis, cystic fibrosis, collagen accumulation in the lungs, broncho-pulmonary dysplasia (BPD); chronic obstructive pulmonary diseases (e.g. emphysema and chronic bronchitis) occupational lung diseases, bronchioliterans organizing pneumonia, pulmonary fibrosis, including idiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis; pulmonary sarcoidosis; and allergies, including allergic rhinitis, contact dermatitis, atopic dermatitis, asthma, asbestosis, coal worker's pneumoconiosis, silicosis or similar conditions associated with long-term exposure to fine particles, and chronic fibrotic lung disease of preterm infants, and ARDS, all of which may be treated with combinations of an IL-1 inhibitor and an IL-4 inhibitor and/or IL-13 inhibitor, e.g. IL-4/13 trap, IL-4R antibody that inhibits IL-13 and IL-4 activity, an anti-IL-13 antibody, or IL-13Ra2-Fc-CTP-Fc.

Such combinations are useful also for treating patients suffering from various skin disorders, including but not limited to dermatitis herpetiformis (Duhring's disease), atopic dermatitis, contact dermatitis, urticaria (including chronic idiopathic urticaria), and autoimmune blistering diseases, including pemphigus vulgaris and bullous pemphigoid. Other diseases treatable with the combination of an IL-1 inhibitor and an IL-4 and/or IL-13 inhibitor include myesthenia gravis, sarcoidosis, including pulmonary sarcoidosis, scleroderma, reactive arthritis, hyper IgE syndrome, multiple sclerosis and idiopathic hypereosinophil syndrome. The combination is used also for treating allergic reactions to medication and as an adjuvant to allergy immunotherapy.

The IL-1R inhibitor and pharmaceutical compositions described herein are useful for treating protozoal diseases, including malaria and schistosomiasis and to treat erythema nodosum leprosum; bacterial or viral meningitis; tuberculosis, including pulmonary tuberculosis; and pneumonitis secondary to a bacterial or viral infection including influenza infection and infectious mononucleosis. Cardiovascular disorders and injuries are treatable and/or preventable with disclosed either pharmaceutical compositions or IL-1 inhibitors alone or in combination with other cytokine inhibitors. Cardiovascular disorders treatable include aortic aneurysms; including abdominal aortic aneurysms, acute coronary syndrome, arteritis; vascular occlusion, including cerebral artery occlusion; complications of coronary by-pass surgery; ischemia/reperfusion injury; heart disease, including atherosclerotic heart disease, myocarditis, including chronic autoimmune myocarditis and viral myocarditis; heart failure, including chronic heart failure, congestive heart failure, cachexia of heart failure; myocardial infarction; restenosis and/or atherosclerosis after heart surgery or after carotid artery balloon angioplastic procedures; silent myocardial ischemia; left ventricular pump dysfunction, post implantation complications of left ventricular assist devices; Raynaud's phenomena; thrombophlebitis; vasculitis, including Kawasaki's vasculitis; veno-occlusive disease, giant cell arteritis, Wegener's granulomatosis; mental confusion following cardio pulmonary by pass surgery, and Schoenlein-Henoch purpura.

Also provided herein are methods for using IL-1 inhibitors of the invention, compositions, and combination therapies to treat various hematologic and oncologic disorders. For example, IL-1 inhibitors, alone or in combination with other cytokine inhibitors or other active agents as described above, can be used to treat various forms of cancer, including acute myelogenous leukemia, chronic myelogenous leukemia leukemia, Epstein-Barr virus-positive nasopharyngeal carcinoma, glioma, colon, stomach, prostate, renal cell, cervical and ovarian cancers, lung cancer (SCLC and NSCLC), including cancer-associated cachexia, fatigue, asthenia, paraneoplastic syndrome of cachexia and hypercalcemia. Solid tumors, including sarcoma, osteosarcoma, and carcinoma, such as adenocarcinoma (for example, breast cancer) and squamous cell carcinoma are also treatable. Additional treatable cancers include esophogeal cancer, gastric cancer, gall bladder carcinoma, leukemia, including acute myelogenous leukemia, chronic myelogenous leukemia, myeloid leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia. Other malignancies with invasive metastatic potential, including multiple myeloma, can be treated with the subject compounds, compositions and combination therapies.

In addition, the disclosed IL-1 inhibitors can be used to treat anemias and hematologic disorders, including chronic idiopathic neutropenia, anemia of chronic disease, aplastic anemia, including Fanconi's aplastic anemia; idiopathic thrombocytopenic purpura (ITP); thrombotic thrombocytopenic purpura, myelodysplastic syndromes (including refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation); myelofibrosis/myeloid metaplasia; and sickle cell vasocclusive crisis.

Various lymphoproliferative disorders also are treatable with IL-1 inhibitors of the invention, including autoimmune lymphoproliferative syndrome (ALPS), chronic lymphoblastic leukemia, hairy cell leukemia, chronic lymphatic leukemia, peripheral T-cell lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, Burkitt's lymphoma, Epstein-Barr virus-positive T cell lymphoma, histiocytic lymphoma, Hodgkin's disease, diffuse aggressive lymphoma, acute lymphatic leukemias, T gamma lymphoproliferative disease, cutaneous B cell lymphoma, cutaneous T cell lymphoma (i.e., mycosis fungoides) and Sezary syndrome.

Non-arthritic disorders of the bones and joints and also treatable with the constructs and compositions described herein. This encompasses osteoclast disorders that lead to bone loss, such as but not limited to osteoporosis, including post-menopausal osteoporosis, osteoarthritis, periodontitis resulting in tooth loosening or loss, and prosthesis loosening after joint replacement (generally associated with an inflammatory response to wear debris). This latter condition also is called “orthopedic implant osteolysis.” Another condition treatable with the compounds, compositions and combination therapies of the invention is temporal mandibular joint dysfunction (TMJ).

The IL-1 inhibitors or pharmaceutical compositions of the invention can also be used to treat rheumatic disorders including adult and juvenile rheumatoid arthritis; scleroderma; systemic lupus erythematosus; gout; osteoarthritis; polymyalgia rheumatica; seronegative spondylarthropathies, including ankylosing spondylitis, and Reiter's disease, psoriatic arthritis and chronic Lyme arthritis. The antibodies of this invention are also useful for treating inflammation of the voluntary muscle and other muscles, including dermatomyositis, inclusion body myositis, polymyositis, and lymphangioleimyomatosis.

Another use for the constructs and pharmaceutical compositions of the invention is the treatment and/or prevention of primary amyloidosis and the secondary amyloidosis that is characteristic of various condition including Alzheimer's disease, secondary reactive amyloidosis; Down's syndrome; and dialysis-associated amyloidosis. Also treatable with the pharmaceutical compositions of the invention are inherited periodic fever syndromes, including familial Mediterranean fever, hyperimmunoglobulin D and periodic fever syndrome and TNF-receptor associated periodic syndromes (TRAPS).

The IL-1-targeting fusion protein of the invention may be used in the treatment of IL-1 related diseases or condition, such as autoinflammatory disorders. One important group of autoinflammatory disorders encompasses autosomal dominant conditions associated with mutations in CIAS-1, a gene that encodes a pyrin-related protein called “cryopyrin” (Feldmann et al. (2002) Am. J. Hum. Genet. 71:198-203; Hoffman et al. (2001) Nat. Genet. 29:301-305). These disorders include Neonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), Muckle-Wells Syndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS). Another condition which may be treated is FMF, a recessively inherited condition characterized by episodes of fever and serositis or synovitis; some subjects also develop systemic amyloidosis (Balow et al. (1997) Genomics 44:280-291). The IL-1 inhibitors of the invention may also be used to treat Still's Disease (systemic onset juvenile idiopathic arthritis), is manifest by spiking fevers, evanescent salmon color rash, arthritis, arthralgia, and hepatosplenomegaly (Masson et al. (1995) Rev. Rhum. Engl. Ed. 62:748-757; Spiegel et al. (2000) Arthritis Rheum. 43:2402-2409). Other diseases that have also been considered autoinflammatory include Kawasaki disease, Blau's syndrome, Early Onset Sarcoidosis (EOS), granulomatosis, arthritis and uveitis., Hidradenitis suppurativa, Behcet's, hyperimmunoglobulinaemia D with periodic fever syndrome (HIDS), tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), and Pyogenic sterile arthritis, pyoderma gangrenosum and acne (PAPA syndrome). IGF-I and IGF II are involved in a number of pathophysiological conditions, including tumorigenesis, and high serum blood levels of IGF-I/IGF-II are associated with risk for development of breast, prostate, colon and lung cancer (LeRoith et al. (2003) Cancer Lett. 195:127-137). In diabetic retinopathy, atherosclerosis and restenosis there is elevated levels of IGF1 and IGF II (Angard et al Artery (1991) 18:197-225; Forrester et al. (1991) Am J Cardiology 68:24C-33C.)

Suitable Subject for Treatment

In one embodiment, a suitable subject for treatment is a human diagnosed as suffering from specific conditions improved by inhibition or reduction of IL-13 include atopic dermatitis, immune complex disease (such as lupus, nephritis, and Grave's disease) allergic conditions, hyper IgE syndrome, immune deficiencies, idiopathic pulmonary fibrosis, hepatic fibrosis, HIV, pulmonary ‘remodeling’, COPD, ulcerative colitis, cancer, Hodgkin's Lymphoma, bullous pemphigoid, transplant and graft vs host disease viral, parasitic, bacterial disease and fungal infection.

In another embodiment, a suitable subject for treatment is a human diagnosed as suffering from a condition which is improved, ameliorated, or inhibited with an IL-1 antagonist, such as familial mediterranean fever (FMF), NOMID/CINCA, Muckle-Wells Syndrome, FCAS, and tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS).

Combination Therapies

In numerous embodiments, the fusion polypeptides of the invention may be administered in combination with one or more additional compounds or therapies. In a preferred embodiment, an IL-13-specific monomer of the invention is co-administered with an agent capable of inhibiting IL-4, such as an anti-IL-4 antibody or an IL-4 mutein. Other combinations include, short-acting inhaled beta2 agonists, oral beta2 agonists, inhaled anticholinergics, oral corticosteroids, inhaled corticosteroids, cromolyn sodium (GASTROCROM™, Celltech), nedocromil, long-acting beta2 agonists, leukotriene modifiers, theophylline, calcinerin inhibitors, picrolimus, sirolimus, anti-IgE (Zolair. Genentech), NFKB inhibitors, p38 MAP kinase inhibitors (VX-702), ICE inhibitors (VX-765), IL-1 inhibitors (IL-1 trap, Regeneron; KINERET™, Amgen), TNFa inhibitors (REMICADE™, Centocor; ENBREL™, Amgen; HUMIRA™, Abbott), IL-5 inhibitors, IL-18 inhibitors, IFN-γ inhibitors, IFN-α blockers. For example, multiple fusion polypeptides can be co-administered, or one or polypeptide can be administered in conjunction with one or more therapeutic compounds. A benefit of the combined use of the fusion polypeptide of the invention with a second therapeutic agent is that it provides improved efficacy and/or reduced toxicity of either therapeutic agent.

In specific embodiments of the therapeutic method of the invention, the subject is treated with a combination of a first IL-1-binding fusion protein trap molecule and a second therapeutic agent. The second therapeutic agent may be a second IL-1 antagonist, such as, for example, a second IL-1-binding fusion protein trap, anakinra (KINERET™, Amgen), a recombinant, nonglycosylated form of the human IL-1 receptor antagonist (IL1Ra), or an anti-IL-18 drug such as IL-18BP or a derivative, an IL-18-binding fusion protein trap (an “IL-18 trap”), anti-IL-18, anti-IL-18R1, or anti-IL-18RAcp antibodies or antibody fragments. Other co-therapies include low dose colchine for FMF, aspirin or other NSAIDs, steroids such as prednisolone, methotrexate, low dose cyclosporine A, TNF inhibitors such as Enbrel®, or Humira®, other inflammatory inhibitors such as inhibitors of caspase-1, p38, IKK1/2, CTLA-4Ig, anti-IL-6 or anti-IL6Ra, etc.

Methods of Administration

The invention provides methods of treatment comprising administering to a subject an effective amount of a fusion polypeptide of the invention. In a preferred aspect, the fusion polypeptide is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably a mammal, and most preferably a human.

Various delivery systems are known and can be used to administer an agent of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction can be enteral or parenteral and include but are not limited to intradermal, intramuscular, intra-articular, intravenous, subcutaneous, intranasal, intraocular, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. Administration can be acute or chronic (e.g. daily, weekly, monthly, etc.) or in combination with other agents. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In another embodiment, the active agent can be delivered in a vesicle, in particular a liposome, in a controlled release system, or in a pump. In another embodiment where the active agent of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see, for example, U.S. Pat. No. 4,980,286), by direct injection, or by use of microparticle bombardment, or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination. Systemic expression may also be achieved by plasmid injection (intradermally or intramuscularly) and electroporation into cells.

A composition useful in practicing the methods of the invention may be a liquid comprising an agent of the invention in solution, in suspension, or both. The term “solution/suspension” refers to a liquid composition where a first portion of the active agent is present in solution and a second portion of the active agent is present in particulate form, in suspension in a liquid matrix. A liquid composition also includes a gel. The liquid composition may be aqueous or in the form of an ointment.

In one embodiment, the pharmaceutical composition of the invention is a sustained release composition. Sustained release formulations for delivery of biologically active peptides are known to the art. For example, U.S. Pat. No. 6,740,634, herein specifically incorporated by reference in its entirety, describes a sustained-release formulation containing a hydroxynaphtoic acid salt of a biologically active substance and a biodegradable polymer. U.S. Pat. No. 6,699,500, herein specifically incorporated by reference in its entirety, discloses a sustained-release formulation capable of releasing a physiologically active substance over a period of at least 5 months.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions comprising a fusion polypeptide of the invention. Such compositions comprise a therapeutically effective amount of one or more fusion polypeptide(s), and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

The fusion polypeptide of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the fusion polypeptide that will be effective for its intended therapeutic use can be determined by standard clinical techniques based on the present description. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. Generally, suitable dosage ranges for intravenous administration are generally about 0.02-10 milligrams active compound per kilogram body weight.

Cellular Transfection and Gene Therapy

The present invention encompasses the use of nucleic acids encoding the fusion polypeptides of the invention for transfection of cells in vitro and in vivo. These nucleic acids can be inserted into any of a number of well-known vectors for transfection of target cells and organisms. The nucleic acids are transfected into cells ex vivo and in vivo, through the interaction of the vector and the target cell facilitated by lipid mixes or electroporation. The compositions are administered (e.g., by injection into a muscle) to a subject in an amount sufficient to elicit a therapeutic response. An amount adequate to accomplish this is defined as “a therapeutically effective dose or amount.”

In another aspect, the invention provides a method of reducing levels of a target ligand in a human or other animal comprising transfecting a cell with a nucleic acid encoding a polypeptide of the invention, wherein the nucleic acid comprises an inducible promoter operably linked to the nucleic acid encoding the polypeptide. For gene therapy procedures in the treatment or prevention of human disease, see for example, Van Brunt (1998) Biotechnology 6:1149-1154.

EXAMPLES

The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Example 1 Construction of Self-Assembling Fusion Polypeptides

Using methods known to the art, monomeric IL-1-specific fusion polypeptides were generated with a His tag, comprising hIL-1RAcp (SEQ ID NO:14), hIL-1RAcp.hIL-1R1myc.myc.His6 (SEQ ID NO:12) or monomeric self-associating fusion polypeptide such as hIL-1RAcp.hIL-1R1.TG.hFc.ARA.CTP.TG.hFc (SEQ ID NO:11), hIL-1RAcp.hIL-1R1.hFc (SEQ ID NO:17), and hIL-1R1.hFc.hIL-1RAcp (SEQ ID NO:9). A second set of IL-13-specific molecules were generated: hIL-13Rα2.hFc (SEQ ID NO:15) and hIL-13Rα2.hFc.CTP.hFc (SEQ ID NO:16). A third set of IGF specific molecules were generated hIGFIR(1-489).hFc.CTP.hFc.CTP (SEQ ID NO:8) and hIGFR(1-489).hFc.CTP (SEQ ID NO:7).

Example 2 In Vitro Bioactivity of Fusion Polypeptides

Self-associating polypeptides were produce as small-scale supernatants by transiently transfecting CHO cells, using Lipofectamine/LIPO plus (Life Technologies), with DNA constructs encoding the proteins. Supernatants were collected after 72 hours and protein expression was measured by Western blotting with anti-human Fc or antibodies against human IL-13ra2 or IL-1R1 HRP-conjugated antibody (Promega) and visualized by ECL (Pierce). Briefly, 5.4×10⁵ CHOK1 cells per well of a 6 well tissue culture dish were transfected using 1 μg of DNA and 5 μl of lipofectamine in OptiMEM™ (Gibco). After 12 h the cells were washed with OptiMEM™ and 2 ml of CHO serum free medium (Gibco) was added. After 60 h and 72 h the media was collected and centrifuged to remove cellular debris and 5 μl of the supernatant was run on a 4-12% Tris Glycine SDS PAGE gel under reducing conditions. The proteins were then transferred to PVDF membranes using standard western blot procedures and incubated with horseradish peroxidase conjugated antibody against human Fc, IL-13Ra2 or IL-1R1, visualized with ECL, and quantified. Bioactivities of the fusion polypeptides generated in Example 1 were tested as follows:

TF1 Bioassay. TF1 cells that had been stably transfected with hIL-13Rα1 were maintained in growth media (10 ng/ml GM-CSF, RPMI 1640, 10% FBS, L-glutamine, penicillin, Streptomycin). For the bioassay, cells are washed 3 times in assay media (as above but without GM-CSF) and then plated at 2×10⁴ cells in 50 μl of assay media. The purified fusion polypeptides were serially diluted into assay media. 25 ul of each of the 13 fusion polypeptides was added to the cells. 25 μl of either IL-13 (15 pM) was then added to the wells containing the cells and the fusion polypeptides. Cells were then incubated at 37° C., 5% CO₂ for ˜70 hrs. The extent of TF1 cell proliferation was measured by the CCK-8 assay according to the manufacturer's protocol (Dojindo Laboratories). The results are shown in Table 1 below.

IL-1 Bioassay. The HEK293/NFκB-luciferase bioassay is used to determine the ability of the IL-1-specific polypeptides of the invention to block the activity of human IL-1 (hIL-1). Human embryonic kidney 293, HEK293, cells, were transfected with an NFκB-luciferase reporter plasmid. By placing an NFκB promoter element upstream of the luciferase gene one can monitor NFκB activity in cells. Because IL-1 signaling is mediated by NFκB, when cells containing the 293/NFκB luciferase construct the luciferase gene is expressed and luciferase activity can be detected in cell lysates. A stable, transfected, cell line, HEK293/D9, was selected for good response to IL-1β as detected by luciferase activity.

For the assay, NFκB-Luciferase cells were suspended at 1.25×10⁵ cells per ml in medium and 0.08 ml of cells plated (10,000 cells per well) into the wells of a 96 well tissue culture plate. Plates were incubated for ˜16 hours at 37° C. in a humidified 5% CO₂ incubator. hIL-1-specific polypeptides and recombinant human IL-1β at varying doses were separately mixed in a 96 well tissue culture dish. 0.026 ml of each of these mixtures were then added to the 96 well plate (hIL-1-specific polypeptides added first) containing the NFκB-Luciferase cells such that the final concentration of IL-1β is 4 pM and the final concentrations of the hIL-1-specific self associating polypeptides ranged from 0.017 pM to 30 nM. Control wells contain no hIL-1-specific polypeptide. Plates were incubated at 37° C. for 6 hours in a humidified 5% CO₂ incubator. After 6 hours, the plates were equilibrated to room temperature for ˜30 minutes and 130 μl of Steady-Glo™ luciferase substrate (Promega) was added. Plates were incubated at room temperature for ˜10 minutes and then read on a Victor multilabel counter (Luminescence 1 sec/well). IC50s were measured which is a 50% reduction in IL-1 stimulated activity, then determined with a 4 parameter fit analysis using Prism software from Graph Pad™.

In vitro binding activity of the fusion polypeptides. Wells were coated with 2 ug/ml of anti-hFc antibody (Pierce) overnight at 4° C. and blocked with PBS with 05% Tween and 10% BSA (PBST). IGF trap molecules were added at different dilutions. After 1 hr at room temperature, the wells were washed 4× with PBST and biotinylated IGF-1 (GroPep) added at 1 ug/ml. Streptavidin HRP (0.2 ug/ml) in PBST was added and developed for 20 min. Kd was determined using standard techniques using Prism software from Graph Pad™.

TABLE 1 IC₅₀ Fusion Polypeptide KD pM hIL-1RAcp.hIL-1R1.myc.myc.His6 3400 (SEQ ID NO: 12) hIL-    23 1RI.TG.hFc.ARA.CTP.TG.hFc.ARA.hIL- 1RAcP (SEQ ID NO: 10) hIL-1RAcp.hIL-1R1  100 TG.hFc.ARA.CTP.TG.hFc (SEQ ID NO: 11) hIL-13Rα2.hFc not (SEQ ID NO: 15) ac- tive hIL-13Rα2.hFc.CTP.hFc Ac- (SEQ ID NO: 16) tive hIGFR (1-489).hFc.CT 1 × 10⁻⁹ M (SEQ ID NO: 7) hIGFR (1-489).hFc.CT.hFc.CT 2 × 10⁻⁹ M (SEQ ID NO: 8)

In order to analyze the expression of the self associated hFc ILI trap, constructs hILIRAcp.hILIR1 (SEQ ID NO:17), hIL-1R1.hFc.hILIRAcp (SEQ ID NO:9), hIL-IRI.TG.hFc.ARA.CTP.TG.hFc.ARA.hIL-IRAcp (SEQ ID NO:10) were expressed in CHOK1 cells. CHOK1 cells were maintained in Ham's F-12 with 10% FBS. Lipofectamine (Gibco) was used to transfect these constructs into CHOK1 cells using standard transient transfection procedures. 5.4×10⁵ CHOK1 cells per well of a 6 well tissue culture dish were transfected using 1 μg of DNA and 5 μl of lipofectamine in optiMEM™ (Gibco). After 12 h the cells were washed with optiMEM and 2 ml of CHO serum free medium (Gibco) was added. After 60 h and 72 h, the media was collected and centrifuged to remove cellular debris and 5 μl of the supernatant was run on a 4-12% Tris Glycine SDS PAGE gel under nonreducing conditions. The proteins were then transferred to PVDF membranes using standard western blot procedures and incubated with horse radish peroxidase conjugated antibody against human Fc (Lane 1=hILIRI.hILIRAcp.hFc (SEQ ID NO:5); lane 2=hILIR1.hFc.hILIRAcp (SEQ ID NO:9); lane 3=hIL R1.hFc.CTP.hFc.hILIRAcP (SEQ ID NO:10)). The results showed that hILIRI.hILIRAcp.hFc (SEQ ID NO:5) and hILIRa.hFc.hILIRAcp both ran at >300 kDa, whereas is hILIRa.hFc.CTP.hFc.hILIRAcP (SEQ ID NO:10) ran at approximately 180 kDa, indicating the hFc are self associating rather than dimerizing as observed in the previous two constructs. 

1. A nucleic acid encoding monomeric fusion polypeptide R1-M-L-M, wherein R1 is a ligand-binding domain of an interleukin receptor capable of binding an interleukin target ligand, M is a self-associating component, and L is a linker, wherein the nucleic acid encodes a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:16.
 2. A nucleic acid encoding monomeric fusion polypeptide R1-M-L-M, wherein R1 is a first ligand-binding domain of an interleukin receptor capable of binding an interleukin target ligand, M is a self-associating component, L is a linker, and further comprising R2, wherein R2 is a second ligand-binding domain of an interleukin receptor which is capable of binding an interleukin target ligand that is the same or different from the interleukin target ligand of R1, wherein the nucleic acid encodes a fusion polypeptide comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:
 11. 3. A vector comprising the nucleic acid of claim
 1. 4. A host-vector system for the production of a fusion polypeptide comprising the vector of claim 3 in a suitable host cell.
 5. The host vector system of claim 4, wherein the cell is a E. coil, B. subtilis, BHK, COS or CHO cell.
 6. A method of producing a fusion polypeptide, comprising growing the cells of the host-vector system of claim 4 under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced.
 7. A monomeric fusion polypeptide R1-M-L-M, wherein R1 is a ligand-binding domain of an interleukin receptor capable of binding an interleukin target ligand, M is a self-associating component, and L is a linker, comprising the amino acid sequence of SEQ ID NO:16.
 8. A monomeric fusion polypeptide R1-M-L-M further comprising R2, wherein R1 is a first ligand-binding domain of an interleukin receptor capable of binding an interleukin target ligand, R2 is a second ligand-binding domain of an interleukin receptor capable of binding an interleukin target ligand, M is a self-associating component, and L is a linker, comprising the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:11.
 9. A vector comprising the nucleic acid molecule of claim
 2. 10. A host-vector system for the production of a fusion polypeptide comprising the vector of claim 9 in a suitable host cell.
 11. The host vector system of claim 10, wherein the cell is a E. coil, B. subtilis, BHK, GOS or CHO cell.
 12. A method of producing a fusion polypeptide, comprising growing the cells of the host-vector system of claim 10 under conditions permitting production of the fusion polypeptide and recovering the fusion polypeptide so produced. 